In this proposal, we hypothesize that Pdx-1 expression alone is not sufficient for transdifferentiation of hepatocytes into functional insulin-producing cells (IPCs), but this selective conversion requires multiple elements including additional genes and an appropriate microenvironment (i.e. hyperglycaemia). Furthermore, we propose that functional IPCs derived from liver cells may be resistant to autoimmune attack. We will test these hypotheses by following two specific aims (SA). SA-1. To determine the role of Pdx-1, Pdx-I-VP16, Pdx-1/Pax4, or Pdx-l-VP16/Pax4 in selective transdifferentiation of freshly isolated hepatocytes into pancreatic endocrine cells. We will use a highly efficient lentiviral vector (LV) system to transduce these genes, singly or in pairs, into primary hepatocytes isolated and cultured from male nonobese diabetic severe combined immunodeficient (NOD-scid) mice. Each transduction will contain the reporter gene, enhanced green fluorescence protein, driven by a rat insulin promoter (RIP-eGFP), to act as tracking insulin gene expression. Using these genetically modified hepatocytes, we will determine the capability of various genes and their combination to convert the hepatocytes into IPCs. We will study the repertoire of gene expression, protein production, and biologic function as indicative of endocrine pancreas. SA-2. To determine whether the liver-derived IPCs can escape autoimmune attack. Because of their potential therapeutic application in-patients with type 1 diabetes mellitus (T1DM), it is critical to determine whether the liver-derived IPCs can maintain their function in an autoimmune model of T1DM. We will transduce freshly isolated hepatocytes from male NOD-scid mice with Pdx-I-VP16 and Pax4 genes in LVs and implant the cells into the diabetic female NOD mice. We will identify any potential autoimmune rejection by observing changes in blood glucose levels, body weight, and subsequent evaluation of lymphocytic infiltrates in the implantation sites. The information derived from these experiments is critical for translating the results of liver to endocrine pancreas transdifferentiation into clinical application for gene and cell based therapies aimed at curing TIDM.